INTRODUCTION

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Cryptococcus neoformans is a pathogenic fungus which most commonly affects the central nervous system and causes fatal meningoencephalitis in AIDS patients (20). This fungus produces a thick extracellular polysaccharide capsule which is a well-recognized virulence factor (15, 22). The predominant capsular polysaccharide of C. neoformans is glucuronoxylomannan (GXM), which consists of an O-acetylated, -1,3-mannose backbone with xylosyl and glucuronosyl side chains. The extent of O-acetylation and xylosyl substitution varies with serotype. The biochemical pathway for synthesis of the polysaccharide capsule, however, is not clear.

Several acapsular mutants have been isolated by classic mutational approaches (3, 27). Molecular cloning by direct complementation of acapsular mutants has resulted in the isolation of three genes, CAP59, CAP60, and CAP64, which are required for capsule formation (2-4). While these genes are not essential for growth in vitro, each has been shown to be required for virulence in the murine model. DNA sequence analysis was not indicative of their biochemical function except that Cap59p and Cap60p contained a putative transmembrane domain and shared sequence similarity at the center of their coding regions. Functional analysis indicated that the transmembrane domain of Cap59p was required for its ability to complement the cap59 acapsular mutant (2). Immunogold electron microscopy revealed the location of Cap60p to be around the nuclear membrane (3).

Difficulties in using histochemical methods to localize gene products in C. neoformans (3, 29) have been encountered. The green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoriae (24) has emerged as a useful marker in studying protein localization in a variety of organisms. The formation of fluorophore appears to be cell autonomous besides the requirement for molecular oxygen (9, 18, 25). Direct visualization of gene expression in individual cells is therefore possible without distortion caused by fixation, sectioning, and staining.

Although the molecular mechanisms of regulation in capsule synthesis are not clear, it has been shown that a homolog of the GPA1 gene encoding the G-protein alpha subunit in the signal transduction pathway influences capsule production in response to iron limitation (1). Recently, another gene involved in the pheromone response signal transduction cascade, STE12, was also found to modulate the expression of several capsule-associated genes, including CAP59, CAP60, and CAP64 (5). The STE12 gene of C. neoformans shares sequence similarity with the Saccharomyces cerevisiae STE12 gene and its homologs, but STE12 exists only in MAT strains of C. neoformans (31). The STE12 gene is required for haploid fruiting on filamentous agar but not for mating. Experimental infections in the murine model suggested that the STE12 gene is important for virulence in C. neoformans (5).

We isolated and characterized a novel capsular gene, CAP10, which may also encode a protein containing a transmembrane domain. CAP10 is required for capsule formation and its deletion abolishes the ability of the fungus to cause fatal infection in mice. Cellular location of the CAP10 gene product was determined by tagging the Cap10p with a new hybrid GFP specific for C. neoformans. In addition, the Escherichia coli -glucuronidase (GUS) gene was used as a reporter to monitor the levels of expression of CAP10 during different stages of growth. The importance of STE12 in regulating CAP10 expression was also demonstrated.

	MATERIALS AND METHODS

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Strains and media. The cDNA library was constructed by J. C. Edman from mRNA of log-phase cells. Table 1 summarizes the strains used in this study. C. neoformans var. neoformans strains B-3501 (MAT) and B-3502 (MATa) have been described previously (19). B-4500 is a wild-type congenic strain of B-4476 (21). B-4500FO2 is a ura5 auxotroph and LP1 is an ade2 ura5 strain derived from B-4500 (3). Strain cap10F2 is an F₂ progeny obtained from a cross between the acapsular mutant, cap10C (3), and a MATa strain. Strain cap10F2FO is a ura5 auxotroph of cap10F2 and was isolated according to the methods described previously (23). TYCC245F1FO is a ura5 auxotroph of ste12 and TYCC259 is a ura5 auxotroph containing GAL7(p)::STE12 (5). YEPD contains 1% yeast extract, 2% Bacto Peptone, and 2% dextrose. Minimal medium (YNB) contains 6.7 g of yeast nitrogen base without amino acids (Difco) and 20 g of glucose per liter. 5-Fluoroorotic acid (5-FOA) medium contains 6.7 g of yeast nitrogen base (Difco), 1 g of 5-FOA, 50 mg of uracil, and 20 g of glucose per liter.

Transformation of C. neoformans. The electroporation method described by Edman and Kwon-Chung (13) was used to transform C. neoformans. TYCC133 is a stable encapsulated transformant of cap10F2FO containing pYCC133 and CIP3 is a stable acapsular transformant of cap10F2FO containing pCIP3. The stable transformants were uracil prototrophs obtained after three transfers on YEPD medium.

Preparation and analysis of nucleic acid and proteins. Genomic DNA isolation and analysis were as described previously (2). Random hexamer priming was used to label the DNA probes to specific activities of >10⁸ dpm/µg (14). Total RNA was isolated by using the FastRNA kit (Bio 101, Vista, Calif.) and poly(A)⁺ RNA was isolated by using the Oligotex mRNA kit (Qiagen, Valencia, Calif.). Northern blot analysis was performed as described previously (6). Following each hybridization, the blot was exposed to PhosphorImager Screen and the CAP10 specific signal, normalized to that of the actin gene, was quantified with ImageQuant 1.1 (Molecular Dynamics). DNA sequencing was performed by the dideoxy-mediated chain-termination method using a Sequenase version 2.0 kit (U.S. Biochemicals, Cleveland, Ohio). Programs of the University of Wisconsin Genetics Computer Group (Madison, Wis.) were used for analysis of nucleic acid sequences (11).

Construction of plasmids. Table 2 summarizes the plasmids used in this study. The URA5-containing plasmid, pCIP3, was obtained from J. C. Edman. The BamHI-EcoRI fragment of pYCC76 (3), which contained the functional ADE2 gene, was cloned into the BamHI-EcoRI site of pBC(KS+) to yield pYCC123. To recover free plasmids from C. neoformans, genomic DNA from encapsulated transformants was digested with NotI, ligated, and transformed into E. coli. Plasmid pYCC125 was one of several plasmids recovered from E. coli which were able to complement the mutation of cap10F2FO. Plasmids pYCC130, pYCC131, and pYCC132 were subclones of pYCC125 in pCIP3 (Fig. 1A).

View larger version (62K): [in this window] [in a new window]   FIG. 1.   Genomic structure of CAP10. (A) Restriction map of CAP10. Overlapping subclones of pYCC125 are depicted. Plasmids were transformed into the acapsular strain cap10F2FO. The capsular phenotypes of the resulting transformants were scored as indicated. Arrow represents transcriptional direction of CAP10 and triangles represent introns. Filled box represents the coding region of CAP10. B, BamHI; Bg, BglII; D, HindIII; M, MscI; N, NsiI; Xb, XbaI. (B) Southern blot analysis. Genomic DNA of B-4500 was digested with different restriction enzymes and fractionated on an 0.8% agarose gel. The DNA blot was hybridized with the 3.3-kb fragment of pYCC133. (C) Chromosomal location of CAP10. The chromosomal DNA was separated by CHEF gel electrophoresis and stained with ethidium bromide (I). The gel-separated chromosomal DNA was transferred to a nylon membrane and hybridized with the CAP10 probe (II). B-3501 (MAT) and B-3502 (MATa) are wild-type strains.

View larger version (23K): [in this window] [in a new window]   FIG. 2.   Deletion of the CAP10 gene. (A) CAP10 locus and gene replacement vector. B, BamHI; Bg, BglII; M, MscI; N, NsiI; S, SmaI; X, XhoI; Xb, XbaI. (B) Southern blot analysis. Genomic DNAs were prepared from the wild-type strain B-4500 and an acapsular strain, TYCC150. DNAs were digested with XhoI and separated in a 0.8% agarose gel. The blot was hybridized with a probe of the 6.0-kb SmaI-NsiI fragment of pYCC150 (I) or the 1.7-kb MscI-NsiI fragment which was deleted from pYCC150 (II). Arrows at 2.0 and 0.8 kb (B-4500; I) indicate the faint signals corresponding to ADE2 hybridization signals.

GUS activity assay. To monitor the GUS activity in different growing stages, transformants of B-4500FOF2 containing pYCC330 were inoculated in 10 ml of minimal medium containing 2% raffinose in 50-ml Falcon tubes and incubated at 30°C with shaking at 200 rpm for 24 h. One milliliter of this culture was then diluted in 9 ml of fresh minimal medium containing 2% glucose and reincubated as described above. Cells were harvested at 5, 10, 15, 25, 45, and 75 h after transfer and GUS activity was assayed as previously described (30). GUS activity was expressed as picomoles of 4-methylumbelliferone produced per minute per 200 µg of protein. To study the effect of overexpression of STE12 on CAP10 expression, TYCC259 was transformed with pYCC330. For galactose induction, cells were inoculated in 10 ml of minimal medium containing 2% raffinose in a 50-ml Falcon tube and incubated at 30°C with shaking at 200 rpm for 24 h. One milliliter of the culture was inoculated into 9 ml of fresh minimal medium containing either 2% glucose or 2% galactose as a carbon source. Cells were harvested after an additional 20-h incubation. To assay GUS activity in cultures of stationary phase, B-4500FO2 and TYCC245F1FO were transformed with pYCC330 separately. Cells were grown in 10 ml of minimal medium containing 2% raffinose for 24 h and 1 ml of this culture was diluted into 9 ml of fresh minimal medium containing 2% glucose. This culture was then grown for an additional 45 h under the same conditions. Cells were harvested and GUS activity was assayed. Six independent transformants from each strain were assayed for GUS activity.

Protein localization. The immunofluorescence and immunogold localization methods were as described previously (3). GFP was visualized by using an Axiovert 100TV microscope (Carl Zeiss, Jena, Germany), and images were recorded using a C5810 color chilled 3CCD camera (Hamamatsu Corporation, Bridgewater, N.J.) and processed using Adobe Photoshop.

Virulence study. Female BALB/c mice (body weight, 20 g) were injected via the lateral tail vein with each yeast strain as described previously (2) and mortality was monitored.

Nucleotide sequence accession number. The GenBank nucleotide accession number for CAP10 is AF144574.

	RESULTS

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Cloning of the CAP10 gene. We have generated 16 acapsular strains by mutagenesis (3). Among the 16 acapsular strains, four were complemented by CAP59, four were complemented by CAP60, and three were complemented by CAP64 (3). From the remaining five strains, one (cap10C) was randomly chosen in an attempt to complement its acapsular phenotype with a library of genomic DNA from B-4500. Several encapsulated transformants of cap10F2FO were obtained after enriching with a two-polymer aqueous-phase system (2). The plasmid responsible for the complementation was recovered and transformed into E. coli. The plasmid, pYCC125, containing a 5.0-kb insert, complemented the acapsular mutation of cap10F2FO but not any other acapsular mutants in our collection (Fig. 1A). The 5-kb insert was subcloned to minimize the region required for complementation. Plasmid pYCC133 containing a 3.3-kb insert was the smallest clone obtained (Fig. 1A). We designated the newly isolated gene CAP10.

Sequence analysis and genomic structure of CAP10. DNA sequences of the genomic and cDNA clones of CAP10 were determined. Because the full-length cDNA was absent in our cDNA library, the 5' portion of the cDNA was obtained by the 5' RACE method. Comparisons of the genomic and cDNA sequences revealed the presence of three introns in CAP10. The presence of multiple introns in genes is a commonly observed feature in C. neoformans. Unlike the other three CAP genes (2-4), no other transcript was detected in close proximity to the CAP10 locus when pYCC125 was used as a probe. The CAP10 gene encodes a putative protein containing 640 amino acids with a calculated molecular mass of 73 kDa. Database searches did not reveal any gene sharing significant similarity with CAP10. However, a putative type II transmembrane region was detected close to the N terminus.

The importance of CAP10 in capsule formation and virulence. A positive-negative selection method was used to delete CAP10 from a wild-type strain (2). This method required a double crossover at the flanking region of the gene. However, the largest clone, pYCC125, which complemented the cap10 mutation contained only 90 bp beyond the stop codon of CAP10 (Fig. 1A). To construct the CAP10 deletion construct, a longer 3' flanking region of CAP10 was obtained by screening an XbaI-digested partial genomic library. The deletion construct, pYCC150, which contained 1.1 kb flanking both 5' and 3' regions of CAP10 is shown in Fig. 2A. This plasmid was used to transform an ade2 ura5 strain, LP1, and the yeast cells were plated on 5-FOA plates. The DNA of acapsular transformants was isolated and analyzed by Southern blotting. Figure 2B shows that the >12-kb band in B-4500 was replaced by an 8.0-kb and a 2.0-kb band in TYCC150, which indicated the replacement of the wild-type CAP10 with the deletion construct. The replacement event was further supported by the lack of hybridization signal in TYCC150 when the blot was hybridized with a probe of the deleted DNA fragment of CAP10 (Fig. 2BII). These results indicated that the acapsular phenotype of the transformant was a result of the CAP10 disruption.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Virulence test. Groups of eight mice each were injected with about 106 viable cells and monitored for 100 days to determine mortality. (A) B-4500, a wild-type strain; TYCC133, a stable Cap+ transformant of cap10F2FO; CIP3, a stable Cap transformant of cap10F2FO harboring only the vector sequence; cap10F2, a cap10 mutant. (B) B-4500FO2, a CAP10 ura5 auxotroph; TYCC150, a ura5 auxotrophic cap10 disruptant.

The influence of different stages of growth on the expression of CAP10. The GUS reporter gene has been successfully used to study the expression of several genes in C. neoformans (5, 30). We constructed a plasmid, pYCC330, in which the coding region of GUS was placed under the control of the CAP10 promoter. This construct was transformed into B4500FO2. GUS activity was measured by using protein extracts from transformants of different stages of growth (see Materials and Methods). Noticeable GUS activity was observed from overnight cultures using raffinose as a carbon source (Fig. 4). When cultures were transferred from raffinose to glucose medium, GUS activity decreased initially and its activity stayed at low levels for 25 h. However, GUS activity increased significantly after prolonged incubation in glucose medium (>45 h). At 3 days after transfer, GUS activity increased about sixfold compared to the activity of 5-h cultures. These results indicated that the expression of the CAP10(p)::GUS was influenced by different growth stages.

View larger version (13K): [in this window] [in a new window]   FIG. 4.   GUS activity of cultures from different growth stages. The GUS reporter construct, pYCC330, was transformed into the wild-type strain B-4500FO2. Protein extracts from three independent transformants were isolated at different time points and the GUS activity was determined. Error bars represent the sample standard deviations.

CAP10 expression is regulated by STE12. Because STE12 of C. neoformans regulates the expression of several virulence-associated genes (5), it was of interest to test whether STE12 regulates the expression of CAP10. Poly(A)⁺ RNA was isolated from 45-h glucose-grown cultures of the ste12 disruptant and the wild-type strain. The mRNA levels of CAP10 were 1.7-fold higher in the wild-type strain than in the ste12 disruptant (Fig. 5A). To test whether the disruption of STE12 affects the CAP10(p)::GUS reporter activity, pYCC330 was transformed into an STE12 strain (B-4500FO2) and ste12 disruptant (TYCC245F1FO). GUS activity was determined from the same stationary-phase culture of both sets of transformants. A significant decrease in GUS activity was observed in transformants of the ste12 disruptant compared to transformants of the wild-type strain (Fig. 5B). Furthermore, to study the effect of overexpression of STE12 on CAP10 expression, pYCC330 was transformed into TYCC259. TYCC259 contains a GAL7(p)::STE12 construct, which can overexpress STE12 when galactose is used as a sole carbon source. GUS activity was measured from protein extracts of glucose- and galactose-grown cultures. GUS activity in galactose-grown culture was slightly higher than that in glucose-grown culture (Fig. 5C). Because the GUS activity in the transformants containing just the vector was also higher in galactose-grown cells than in glucose-grown cells (Fig. 5C), it was possible that the observed differences in GUS activity were caused by differences in culture medium and were not induced by overexpression of STE12. To evaluate this possibility, GUS activity from transformants of a wild-type strain (B-4500FO) containing pYCC330 grown in both culture media was determined. No significant differences in GUS activity were observed between glucose- and galactose-grown cultures for transformants of B-4500FO containing pYCC330 (glucose, 8.00 ± 1.56, versus galactose, 7.37 ± 2.16). Thus, the differences of GUS activity in transformants of TYCC259 were caused by overexpression of STE12, which induced the CAP10(p)::GUS reporter gene activity. Therefore, the observed changes of GUS activity in transformants containing the vector may be due to the existence of a cryptic promoter in the vector. These data indicated that STE12 modulates the expression of CAP10.

View larger version (10K): [in this window] [in a new window]   FIG. 5.   STE12 modulates CAP10 expression. (A) Northern blot analysis. Poly(A)+ RNA isolated from the wild-type strain (B-4500) and the ste12 disruptant (TYCC245F1) was fractionated in a 2.2 M formaldehyde-1% agarose gel and transferred to a nylon membrane. The blot was hybridized with a probe of CAP10 cDNA and a probe of actin cDNA. The phosphorimaging results were used to determine the relative signal intensities. (B) The effect of the deletion of STE12 on GUS reporter activity. (C) The effect of STE12 overexpression on GUS reporter activity. CAP10 denotes the transformants containing pYCC330 and vector denotes the transformants containing the promoterless GUS (pYCC331). Data are averages of GUS activity from six independent transformants. Error bars represent the standard deviations.

Localization of Cap10p. To understand the function of CAP10, we attempted to determine the cellular location of the CAP10 gene product by peptide epitope-tagging methods. Nine amino acids of the influenza virus HA protein were inserted into the C terminus of CAP10, and the resulting construct was transformed into a cap10 strain. The resulting transformants produced capsule, indicating that insertion of the HA tag at the C terminus of Cap10p did not interfere with its function. Total proteins were extracted from these capsule-containing transformants and analyzed by Western blotting using an anti-HA antibody. The size of the protein detected by Western blotting corroborated the predicted molecular weight (Fig. 6I). Immunofluorescence and immunoelectron microscopy were used to determine the cellular location of Cap10p. However, we did not obtain satisfactory results due to technical difficulties, such as suboptimal levels of fluorescence and poor preservation of organelles in immunogold electron microscopy studies (data not shown). Similar negative results were obtained even when three tandem copies of HA were used to tag Cap10p.

View larger version (113K): [in this window] [in a new window]   FIG. 6.   Detection and localization of Cap10p. (I) Immunoblot analysis. Yeast cells were grown in YNB and total protein extracts were analyzed by sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis, incubated with anti-HA antibody, and analyzed using the Western-Star chemiluminescence detection system. Lane 1, a wild-type strain, B-4500; lane 2, an encapsulated transformant of TYCC150 containing pYCC152. (II) Localization of Cap10p-GFP fusion protein. Yeast cells were grown on YNB medium for no more than 24 h at 30°C. Panels A and B show the transformants of TYCC150 containing Cap10p-GFP fusion construct, pYCC352. Panels C and D show the transformants of B-4500FO2 containing pCIP3. Shown are phase-contrast micrographs (A and C) and fluorescence micrographs (B and D).

	DISCUSSION

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Cloning and characterization of CAP10 revealed that the gene contains three introns and encodes a novel protein. CAP10 was not contiguous with another transcript in close proximity. This observation was different from those obtained with the other three CAP genes which are tightly linked with other genes: CAP59 with L27, CAP60 with CEL1, and CAP64 with PRE1 (2-4). Animal studies demonstrated that cap10 mutants constructed by deletion or mutagenesis were unable to produce fatal infection in mice, as demonstrated with other acapsular strains of cap59, cap60, and cap64 (2-4). Complementation of the cap10 mutation restored capsule and virulence. Thus, CAP10 is the fourth characterized gene required for capsule formation and virulence in C. neoformans.

The GFP-tagged Cap10p appeared as patches within the cytoplasm of yeast cells. Because of the presence of a putative type II transmembrane region close to the N terminus, we speculate that Cap10p may be associated with certain types of organelles, although insertion of GFP may have affected the location. We used HA epitope-tagging and immunoelectron microscopy to further define the location of Cap10p without satisfactory results. Similar difficulties have been encountered using histochemical methods to localize gene products in C. neoformans (3, 29). Raising high-titer antibodies against Cap10p may increase the sensitivity of detecting the protein and reveal the definite location of Cap10p.

We have previously used several versions of modified GFP, including yGFP3, to tag Cap10p, but without success. The yGFP3 was used as a reporter by fusing it to different promoters of C. neoformans (10). It is not clear why the CAP10-yGFP3 fusion construct failed to produce strong fluorescence. The only GFP construct that yielded satisfactory results was a hybrid GFP, pYCC352. This hybrid protein contained a portion of yGFP3 engineered for C. albicans at the N terminus and a portion of GFP designed for the plant system at the C terminus. The success of expressing this hybrid GFP in C. neoformans may be due to combinative effects: the removal of the cryptic intron from a thermotolerant GFP mutant (17, 26) and introducing modified chromophore region to increase the fluorescence intensity (7, 8). This hybrid GFP was also successfully used to localize the Cap60p (data not shown) which, by immunogold electron microscopy, has been localized to the nuclear membrane (3). Several factors influenced the results of our hybrid GFP expression. When the promoter of CAP10 in the GFP fusion construct was replaced with a strong inducible GAL7 promoter of C. neoformans (30), the resulting construct was able to complement the acapsular mutation of TYCC150 on galactose medium. However, no GFP signal was detected in these encapsulated transformants from galactose-grown culture (data not shown). Thus, overexpression of fusion GFP showed an adverse effect on the GFP fluorescence signal. Physiological conditions of yeast cells also affected the level of GFP signals. GFP fluorescence was reliably detected only when cultures were grown on agar for no more than 24 h. When older cultures were used, not only did the GFP signals fade but also many yeast cells showed copious autofluorescence. Therefore, it may be important to have appropriate expression levels of the fusion construct for detection of GFP signals in C. neoformans. The expression levels of the fusion construct, however, appeared to have no effect on its function to complement the acapsular mutation.

Using the GUS gene as a reporter system, we found that CAP10 expression is influenced by different stages of growth; the CAP10 gene was expressed at much higher levels during the late stationary phase. It appears that there is a basal level of expression of CAP10 in young cultures and the expression of CAP10 increases when the nutrient of the medium is depleted. These data appear to corroborate the observations that yeast cells produce abundant capsule in late stationary phase (16), although it is not clear how this process is regulated. Interestingly, GUS activity and accumulation of CAP10 mRNA decreased in a strain containing a deletion of a well-conserved transcriptional factor, STE12. In addition, the CAP10(p)::GUS reporter activity was induced by overexpression of STE12. Thus, CAP10 expression is modulated by STE12. Since STE12 is present only in MAT cells, it would be of interest to know what transcriptional factor(s) controls the expression of CAP10 in MATa strains. STE12 is a global regulator, which also controls the expression of several genes involved in virulence, such as capsule and phenol oxidase production (5). Although four capsule-associated genes have been isolated, the regulation of expression of these genes is not well defined. Further investigation on the mechanisms of regulating age-dependent CAP10 expression and how STE12 modulates CAP10 expression may lead to further understanding of the regulation of capsule synthesis.